The invention relates to molecular separation technology and, more particularly, to a robotic instrument for analysis of multiple samples in microchannels.
In the past ten years or so, parallel capillaries have been used extensively for molecular separations, such as by means of electrophoresis. Capillary electrophoresis has been used for the analysis of DNA and proteins, and for the separation of small ions, small molecules, bacteria, and viruses. Different separation media have been used in the capillaries including solutions, gels, and polymers. In each technique, the mobility of the target can be measured.
Capillaries have been applied both to DNA fragment length analysis and to DNA sequencing. The study of nucleotide sequences relies upon the high resolution separation of polynucleotide fragments. Each fragment in a family of fragments is tagged with fluorescent markers and the differences in the molecular migration in a capillary channel are observed. Fragments having differences of only a single base pair are routinely separated with fluorescent detection.
To increase the throughput, many capillaries can be used in parallel. Parallel channel electrophoresis allows many samples to be analyzed simultaneously and can result in high throughput rates.
Recently, several groups have implemented capillary electrophoresis in microchannel formats (A. T. Wooley, G. F. Sensabaugh and R. A. Mathies, xe2x80x9cHigh-Speed DNA Genotyping Using Microfabricated Capillary Array Electrophoresis Chipsxe2x80x9d, Anal. Chem., 69:2181-2186 (1997); A. T. Woolley and R. A Mathies, Anal. Chem., 67:3676-3680 (1995); A. T. Woolley, P. C. Simpson, S. Liu, R. Johnston, G. F. Sensabaugh, A. N. Glazer, and R. A. Mathies, xe2x80x9cAdvances in Microfabricated Integrated DNA Analysis Systemsxe2x80x9d, HPCE98 (1998); P. C. Simpson, D. Roach, A. T. Woolley, T. Thorsen, R. Johnston, G. F. Sensabaugh, and R. A. Mathies, xe2x80x9cHigh-throughput genetic analysis using microfabricated 96-sample capillary array electrophoresis microplatesxe2x80x9d, Proc. Natl. Acad. Sci. USA, 95:2256-2261 (1998). This approach uses microchannels etched or molded into a substrate as the separation channels in place of capillaries (R. M. McCormick, R. Nelson, M. G. Alonso-Amigo, D. J. Benvegnu and H. H. Hooper, xe2x80x9cMicrochannel electrophoretic separation of DNA in injection-molded plastic substratesxe2x80x9d, Anal. Chem. 69:2626-2630 (1997); U.S. Pat. No. 5,376,252, issued 1994 to B. Ekstrom, G. Jacobson, O. Ohman and H. Sjodin). The resulting device is commonly called a microchip, even though the physical size of the entire substrate can vary from microchip size, i.e. dimensions of a few millimeters on a side, to wafer size, i.e. dimensions similar to semiconductor wafers (10-20 centimeters diameter) to microchannels in 48 cm long xe2x80x9cmacrochipsxe2x80x9d (C. Davidson, J. Balch, L. Brewer, J. Kimbrough, S. Swierkowski, D. Nelson, R. Madabhushi, R. Pastrone, A. Lee, P. McCready, A. Adamson, R. Bruce, R. Mariella and A. Carrano, xe2x80x9cDevelopment of a Microchannel Based DNA Sequencerxe2x80x9d, DOE Human Genome Program Contractor-Grantee Workshop VI, Santa Fe, NM (1997)). The determining factors in microchip size are the complexity of microchannel routes and the lengths of the separation channels. The length of the channels must allow for sample input, sample migration and a measurement zone. The channels are typically of dimensions from 8 to 40 micrometers deep and 30 to 150 micrometers wide. The small channels resolve DNA fragments in significantly shorter times than capillaries with larger cross-sectional area.
Beyond providing parallel capillaries, some advances in speed of analysis have been achieved by providing parallel sample wells and providing automated optical detectors and software analyzers. In spite of these advances, fine separations are still a time consuming and labor intensive process, particularly in handling and presentation of the specimen to an analysis instrument.
An object of the invention was to devise an apparatus for automatic handling and presentation of specimens into microchips and macrochips for parallel high throughput analysis in microchannels. A further object was to automate the presentation of the chips to an analysis station where electrodes would be docked, samples injected into the separation channels, the separations performed, and the samples detected.
The above object has been achieved with a macro to micro interface for loading, handling, running, and analyzing samples in an instrument based upon electrophoresis in microchannels on a microchip. The microchip has macroscale inlet ports leading to the microchannels. The inlet ports are spaced apart to match the size and spacings of pipettors in an array of ganged pipettor tips.
The microchannels provide microscopic volumes, much less than a microliter, in which analysis is carried out. The instrument features a microchip handler, with relative motion of the microchip with respect to a pipettor, electrodes, and detector. In some instances the microchip is moved, while in other instances, the other components are moved. There is a sequence of automatic operations involving placing a sample-free microchip on a chuck, loading samples with a pipetting device into the microchip, contacting microchannels in the microchip with electrodes, injecting samples into the separation microchannels, running an electrophoretic separation, detecting and measuring the separation, and then removing the microchip.
In a preferred embodiment, a microchip, prefilled with separation matrix but not sample, is held in a vacuum chuck which is movable with high precision on a first Y-axis track from a sample loading station to a sample analysis station. The microchannels of the microchip are filled beforehand with a polymer or other matrix that may act as a sieve to enhance sample separation. At the sample loading station, samples can be loaded into the microchip by a multifunctional device, that includes a pipettor. The multifunctional device moves along a transverse X-axis gantry between the sample loading station on the first Y-axis track and tip and sample stations both on a second Y-axis track, parallel to the first Y-axis track. The second track can move pipette tips, reagent trays, microtiter trays containing samples, or other objects automatically into position for use by the multifunctional device. The multifunctional device, carried by the gantry, moves up and down on a Z-axis, perpendicular to the X and Y axes. Motion along all axes is driven by stepper motors so that precise and accurate positioning may be achieved. A servo motor or other actuator systems may be used for precise position control.
The multifunctional device contains a plurality of ganged pipettors, an individual pipettor, and a vacuuming line. The plurality of pipettors is ganged with spacings matching the well spacings on a microtiter plate. The same spacings are used for sample loading inlet ports on the microchip. In this manner, a multiple-channel pipettor can simultaneously load multiple samples into sample inlet ports.
The multifunctional device can be moved initially from the sample loading station on the first track to the tip and sample stations on the second track where new pipette tips are applied to the ganged pipettors. The multifunctional device then moves on the gantry to pickup a tip guide and then moves back to the tip and sample stations on the second track. The second track can then be moved to a position where the ganged pipettors on the gantry can withdraw samples from a microtiter plate on the track. The multifunctional device then moves on the gantry to the sample loading station where it deposits the samples into sample inlet ports in the microchip on the first track. The multifunctional device moves back along the gantry first to release the tip guide and then to the tip and sample station where the used tips are discarded into a used tip tray that has been moved into position below the multifunctional device by the second track. The cycle of picking up tips, tip guide, and samples; delivering the sample to the microchip; and then parking the tip guide and discarding the used tips is repeated until the microchip has been completely loaded.
After the microchip has been loaded, it is moved to the sample analysis station on the first track below a sample analysis detector and raised to dock with the array of wire electrodes supported by a platform over the first track. The final position of the microchip places the microchannels in the focal plane of a detector at the sample analysis station. The detector preferably includes a scanning confocal laser microscope capable of detecting fluorescently tagged molecules during separation.
The electrical potential of the electrodes can be controlled to first move precise sub-microliter volumes of the samples from the loading wells into an injection region of the separation microchannels, and then to stimulate electromigration in the separation microchannels.
As the samples separate in the microchannels, a region of the microchip is monitored, typically by a scanning confocal laser microscope to detect the molecular separations. For DNA sequencing, four fluorescent markers are usually detected for forming four-color electropherograms of the separations. The four-color electropherograms can be processed to ultimately call the bases and determine the DNA sequence of the samples.